These investigations seek to identify the molecular mechanisms of microRNA-mediated gene regulation. The isolation and characterization of Drosophila melanogaster mutants will be carried out to identify genes that function in the miRNA pathway and to elucidate the mechanism by which miRNA effector complexes act on target mRNAs. miRNA function impacts public health, given that many human tumors show altered miRNA profiles. In addition, the responsible use of RNA-based therapeutics requires an understanding of miRNA function, given the observation of crosstalk between RNA-mediated silencing pathways. 1. To isolate Drosophila melanogaster mutants with disrupted miRNA-mediated gene regulation 1.1 Generate EMS-mutagenized Drosophila lines with altered expression of a sensor of miRNA function 1.2 Conduct a pilot reverse genetic screen to identify transgeneic lines in the Drosophila RNAi library with altered expression of a sensor of miRNA function 2. To prioritize candidate mutant lines for further study by characterizing additional phenotypic defects 2.1 Analyze candidate lines using alternate sensors of miRNA function to determine whether mutant lines demonstrate general or reporter-specific effects 2.2 Classify candidate lines by comparing the phenotypes of newly isolated mutants to the characterized phenotypes (including developmental and RNAi defects) of known miRNA pathway mutants 3. To identify genes in the miRNA pathway and assess their role in generating miRNA effector complexes 3.1 Characterize alleles of known miRNA pathway genes Dicer-1 and Pasha 3.2 Determine the identities of 2-3 novel genes in the miRNA pathway 3.3 Perform a biochemical evaluation of miRNA complexes to characterize miRNA pathway genes Precise gene regulation is essential to normal biological function. An understanding the newly discovered roles of RNA in gene regulation is essential to our potential ability to combat many gene regulation diseases, most notably cancer.